Striatal bilateral control of skilled forelimb movement.

Skilled motor behavior requires bihemispheric coordination, and participation of striatal outputs originating from two neuronal groups identified by distinctive expression of D1 or D2 dopamine receptors. We trained mice to reach for and grasp a single food pellet and determined how the output pathways differently affected forelimb trajectory and task efficiency. We found that inhibition and excitation of D1-expressing spiny projection neurons (D1SPNs) have a similar effect on kinematics results, as if excitation and inhibition disrupt the whole ensemble dynamics and not exclusively one kind of output. In contrast, D2SPNs participate in control of target accuracy. Further, ex vivo electrophysiological comparison of naive mice and mice exposed to the task showed stronger striatal neuronal connectivity for ipsilateral D1 and contralateral D2 neurons in relation to the paw used. In summary, while the output pathways work together to smoothly execute skill movements, practice of the movement itself changes synaptic patterns.

Growth conditions and survival kinetics during storage of Lactobacillus rhamnosus GG for the design of a sustainable probiotic whey-based beverage containing Costa Rican guava fruit pulp.

The finding of economical and practical applications for milk whey is still a challenge for dairy industries. This paper presents information about the development of a probiotic-prebiotic beverage based on Lactobacillus rhamnosus GG (LGG) and Costa Rican guava (CRG) fruit pulp with industrial potential. First, a supplemented whey media was developed for LGG growth, and the whey-supplemented media was used for fermentation in bioreactors. LGG reached a maximum growth rate of 0.32 hr-1 after 48 hr of fermentation. The whey-grown probiotics were then mixed with CRG pulp to produce the probiotic-prebiotic beverage. The survival kinetics of LGG in the formulated drink was not affected by the addition of CRG pulp (P > 0.05), and the shelf-life of the inoculated beverage surpassed 40 days with a minimum population of 106 colony forming units (CFU)/mL. Properties as pH, fructose, glucose, sucrose, and proanthocyanidins (PACs) content exhibited a significant difference after storage time (P < 0.05). Finally, three different formulas of the beverage with different whey content were compared through sensory evaluation. The prototype with 50% whey content was one of the most valuable beverage formulas according to the organoleptic parameters, which remarks about the possibility of developing a probiotic whey-based beverage containing CRG pulp. Furthermore, this is the first report about CRG beverages as a probiotic vector. PRACTICAL APPLICATION: This research focuses on the evaluation of the properties of a probiotic beverage, with a promissory industrial application using whey, as a dairy industry byproduct, combined with the pulp of the highly nutritious and subutilized Costa Rican guava (CRG) fruit.

Synchronized activation of striatal direct and indirect pathways underlies the behavior in unilateral dopamine-depleted mice.

For more than three decades it has been known, that striatal neurons become hyperactive after the loss of dopamine input, but the involvement of dopamine (DA) D1- or D2-receptor-expressing neurons has only been demonstrated indirectly. By recording neuronal activity using fluorescent calcium indicators in D1 or D2 eGFP-expressing mice, we showed that following dopamine depletion, both types of striatal output neurons are involved in the large increase in neuronal activity generating a characteristic cell assembly of particular neurons that dominate the pattern. When we expressed channelrhodopsin in all the output neurons, light activation in freely moving animals, caused turning like that following dopamine loss. However, if the light stimulation was patterned in pulses the animals circled in the other direction. To explore the neuronal participation during this stimulation we infected normal mice with channelrhodopsin and calcium indicator in striatal output neurons. In slices made from these animals, continuous light stimulation for 15 s induced many cells to be active together and a particular dominant group of neurons, whereas light in patterned pulses activated fewer cells in more variable groups. These results suggest that the simultaneous activity of a large dominant group of striatal output neurons is intimately associated with parkinsonian symptoms.

Cholinergic modulation of striatal microcircuits.

The purpose of this review is to bridge the gap between earlier literature on striatal cholinergic interneurons and mechanisms of microcircuit interaction demonstrated with the use of newly available tools. It is well known that the main source of the high level of acetylcholine in the striatum, compared to other brain regions, is the cholinergic interneurons. These interneurons provide an extensive local innervation that suggests they may be a key modulator of striatal microcircuits. Supporting this idea requires the consideration of functional properties of these interneurons, their influence on medium spiny neurons, other interneurons, and interactions with other synaptic regulators. Here, we underline the effects of intrastriatal and extrastriatal afferents onto cholinergic interneurons and discuss the activation of pre- and postsynaptic muscarinic and nicotinic receptors that participate in the modulation of intrastriatal neuronal interactions. We further address recent findings about corelease of other transmitters in cholinergic interneurons and actions of these interneurons in striosome and matrix compartments. In addition, we summarize recent evidence on acetylcholine-mediated striatal synaptic plasticity and propose roles for cholinergic interneurons in normal striatal physiology. A short examination of their role in neurological disorders such as Parkinson's, Huntington's, and Tourette's pathologies and dystonia is also included.

Thalamic afferents to prefrontal cortices from ventral motor nuclei in decision-making.

The focus of this literature review is on the three interacting brain areas that participate in decision-making: basal ganglia, ventral motor thalamic nuclei, and medial prefrontal cortex, with an emphasis on the participation of the ventromedial and ventral anterior motor thalamic nuclei in prefrontal cortical function. Apart from a defining input from the mediodorsal thalamus, the prefrontal cortex receives inputs from ventral motor thalamic nuclei that combine to mediate typical prefrontal functions such as associative learning, action selection, and decision-making. Motor, somatosensory and medial prefrontal cortices are mainly contacted in layer 1 by the ventral motor thalamic nuclei and in layer 3 by thalamocortical input from mediodorsal thalamus. We will review anatomical, electrophysiological, and behavioral evidence for the proposed participation of ventral motor thalamic nuclei and medial prefrontal cortex in rat and mouse motor decision-making.

Are the Symptoms of Parkinsonism Cortical in Origin?

We present three reasons to suspect that the major deleterious consequence of dopamine loss from the striatum is a cortical malfunction. We suggest that it is cortex, rather than striatum, that should be considered as the source of the debilitating symptoms of Parkinson's disease (PD) since:1.Cortical synapses onto striatal dendritic spines are lost in PD.2.All known treatments of the symptoms of PD disrupt beta oscillations. Oscillations that are also disrupted following antidromic activation of cortical neurons.3.The final output of basal ganglia directly modulates thalamic connections to layer I of frontal cortical areas, regions intimately associated with motor behaviour. These three reasons combined with evidence that the current summary diagram of the basal ganglia involvement in PD is imprecise at best, suggest that a re-orientation of the treatment strategies towards cortical, rather than striatal malfunction, is overdue.

Presynaptic D1 heteroreceptors and mGlu autoreceptors act at individual cortical release sites to modify glutamate release.

The aim of this work was to study release of glutamic acid (GLU) from one-axon terminal or bouton at-a-time using cortical neurons grown in vitro to study the effect of presynaptic auto- and heteroreceptor stimulation. Neurons were infected with release reporters SypHx2 or iGluSnFR at 7 or 3 days-in-vitro (DIV) respectively. At 13-15 DIV single synaptic boutons were identified from images obtained from a confocal scanning microscope before and after field electrical stimulation. We further stimulated release by raising intracellular levels of cAMP with forskolin (10µM). Forskolin-mediated effects were dependent on protein kinase A (PKA) and did not result from an increase in endocytosis, but rather from an increase in the size of the vesicle readily releasable pool. Once iGluSnFR was confirmed as more sensitive than SypHx2, it was used to study the participation of presynaptic auto- and heteroreceptors on GLU release. Although most receptor agonizts (carbamylcholine, nicotine, dopamine D2, BDNF) did not affect electrically stimulated GLU release, a significant increase was observed in the presence of metabotropic D1/D5 heteroreceptor agonist (SKF38393 10µM) that was reversed by PKA inhibitors. Interestingly, stimulation of group II metabotropic mGLU2/3 autoreceptors (LY379268 50nM) induced a decrease in GLU release that was reversed by the specific mGLU2/3 receptor antagonist (LY341495 1µM) and also by PKA inhibitors (KT5720 200nM and PKI14-22 400nM). These changes in release probability at individual release sites suggest another level of control of the distribution of transmitter substances in cortical tissue.

The neostriatum: two entities, one structure?

The striosome (or patch) was first identified with anatomical techniques as neurons organized in a three-dimensional labyrinth inserted in and interdigitating the rest of neostriatum: the matrix. Striosome and matrix rapidly became known as two neuronal compartments expressing different biochemical markers, embryonic development and afferent and efferent connectivity. In spite of extensive intrinsic neuronal axonal and dendritic extensions supposed to exchange information between matrix and striosomes, evidence suggested the presence of independent areas. Here, we report that indeed these two areas do not exchange synaptic information. We used genetic expression of channel rhodopsin 2 carried by adeno-associated virus serotype 10 (AAVrh10) that only expresses in neurons of the matrix compartment. Whole-cell patch-clamp recordings of matrix neurons activated by light pulses consistently produced inhibitory postsynaptic currents (IPSCs), but the same manipulation did not evoke IPSCs in striosome neurons. The matrix contains both direct and indirect striatal output pathways. By targeting striatal matrix expression of designer receptors exclusively activated by a designer drug (DREADD) hM3di carried by AAVrh10, we were able to inhibit the matrix neuronal compartment of the dorsolateral striatum during performance of a learned single-pellet reach-to-grasp task. As expected, inhibition of matrix neurons by systemic administration of DREADD agonist clozapine-n-oxide interfered with performance of the learned task.

Basal ganglia-thalamus and the "crowning enigma".

When Hubel (1982) referred to layer 1 of primary visual cortex as "… a 'crowning mystery' to keep area-17 physiologists busy for years to come …" he could have been talking about any cortical area. In the 80's and 90's there were no methods to examine this neuropile on the surface of the cortex: a tangled web of axons and dendrites from a variety of different places with unknown specificities and doubtful connections to the cortical output neurons some hundreds of microns below. Recently, three changes have made the crowning enigma less of an impossible mission: the clear presence of neurons in layer 1 (L1), the active conduction of voltage along apical dendrites and optogenetic methods that might allow us to look at one source of input at a time. For all of those reasons alone, it seems it is time to take seriously the function of L1. The functional properties of this layer will need to wait for more experiments but already L1 cells are GAD67 positive, i.e., inhibitory! They could reverse the sign of the thalamic glutamate (GLU) input for the entire cortex. It is at least possible that in the near future normal activity of individual sources of L1 could be detected using genetic tools. We are at the outset of important times in the exploration of thalamic functions and perhaps the solution to the crowning enigma is within sight. Our review looks forward to that solution from the solid basis of the anatomy of the basal ganglia output to motor thalamus. We will focus on L1, its afferents, intrinsic neurons and its influence on responses of pyramidal neurons in layers 2/3 and 5. Since L1 is present in the whole cortex we will provide a general overview considering evidence mainly from the somatosensory (S1) cortex before focusing on motor cortex.

Cell Assembly Signatures Defined by Short-Term Synaptic Plasticity in Cortical Networks.

The cell assembly (CA) hypothesis has been used as a conceptual framework to explain how groups of neurons form memories. CAs are defined as neuronal pools with synchronous, recurrent and sequential activity patterns. However, neuronal interactions and synaptic properties that define CAs signatures have been difficult to examine because identities and locations of assembly members are usually unknown. In order to study synaptic properties that define CAs, we used optical and electrophysiological approaches to record activity of identified neurons in mouse cortical cultures. Population analysis and graph theory techniques allowed us to find sequential patterns that represent repetitive transitions between network states. Whole cell pair recordings of neurons participating in repeated sequences demonstrated that synchrony is exhibited by groups of neurons with strong synaptic connectivity (concomitant firing) showing short-term synaptic depression (STD), whereas alternation (sequential firing) is seen in groups of neurons with weaker synaptic connections showing short-term synaptic facilitation (STF). Decreasing synaptic weights of a network promoted the generation of sequential activity patterns, whereas increasing synaptic weights restricted state transitions. Thus in simple cortical networks of real neurons, basic signatures of CAs, the properties that underlie perception and memory in Hebb's original description, are already present.

Rebuilding a realistic corticostriatal "social network" from dissociated cells.

Many of the methods available for the study of cortical influences on striatal neurons have serious problems. In vivo the connectivity is so complex that the study of input from an individual cortical neuron to a single striatal cell is nearly impossible. Mixed corticostriatal cultures develop many connections from striatal cells to cortical cells, in striking contrast to the fact that only connections from cortical cells to striatal cells are present in vivo. Furthermore, interneuron populations are over-represented in organotypic cultures. For these reasons, we have developed a method for growing cortical and striatal neurons in separated compartments that allows cortical neurons to innervate striatal cells in culture. The method works equally well for acutely dissociated or cryopreserved neurons and allows a number of manipulations that are not otherwise possible. Either cortical or striatal compartments can be transfected with channel rhodopsins. The activity of both areas can be recorded in multielectrode arrays or individual patch recordings from pairs of cells. Finally, corticostriatal connections can be severed acutely. This procedure enables determination of the importance of corticostriatal interaction in the resting pattern of activity. These cultures also facilitate development of sensitive analytical network methods to track connectivity.

Extrasynaptic glutamate NMDA receptors: key players in striatal function.

N-methyl-D-aspartate receptors (NMDAR) are crucial for the function of excitatory neurotransmission and are present at the synapse and on the extrasynaptic membrane. The major nucleus of the basal ganglia, striatum, receives a large glutamatergic excitatory input carrying information about movements and associated sensory stimulation for its proper function. Such bombardment of glutamate synaptic release results in a large extracellular concentration of glutamate that can overcome the neuronal and glial uptake homeostatic systems therefore allowing the stimulation of extrasynaptic glutamate receptors. Here we have studied the participation of their extrasynaptic type in cortically evoked responses or in the presence of NMDARs stimulation. We report that extrasynaptic NMDAR blocker memantine, reduced in a dose-dependent manner cortically induced NMDA excitatory currents in striatal neurons (recorded in zero-Mg(++) plus DNQX 10 µM). Moreover, memantine (2-4 µM) significantly reduced the NMDAR-dependent membrane potential oscillations called up and down states. Recordings of neuronal striatal networks with a fluorescent calcium indicator or with multielectrode arrays (MEA) also showed that memantine reduced in a dose-dependent manner, NMDA-induced excitatory currents and network behavior. We used multielectrode arrays (MEA) to grow segregated cortical and striatal neurons. Once synaptic contacts were developed (>21DIV) recordings of extracellular activity confirmed the cortical drive of spontaneous synchronous discharges in both compartments. After severing connections between compartments, active striatal neurons in the presence of memantine (1 µM) and CNQX (10 µM) were predominantly fast spiking interneurons (FSI). The significance of extrasynaptic receptors in the regulation of striatal function and neuronal network activity is evident.

Development of dissociated cryopreserved rat cortical neurons in vitro.

Dissociated neuronal cultures of various brain regions are commonly used to study physiological and pathophysiological processes in vitro. The data derived from these studies are often viewed to have relevance to processes taking place in the mature brain. However, due to the practical challenges associated with lengthy neuronal culture, neurons are often kept for 14 days in vitro (DIV), or less, before being subject to experimentation. Non-proliferative cultures such as primary neuronal cultures can be maintained for more than 42 DIV if water evaporation from culture media is monitored and corrected. To determine appropriate time points corresponding to the stages of cortical development, we compared characteristics of cryopreserved cortical neurons in cultures at various DIV using immunofluorescence, biochemical measurements and multielectrode array recordings. Compared to 21 and 35 DIV, at 14 DIV, cultures are still undergoing developmental changes and are not representative of adult in vivo brain tissue. Specifically, we noted significant lack in immunoreactivity for synaptic markers such as synapsin, vesicular GABA transporter and vesicular glutamate transporter at 14 DIV, relative to 21 and 35 DIV. Moreover, multielectrode array analysis indicated an increase in network firing up to 46 DIV with patterned firing peaking at 35 DIV. Our results provide specific evidence of the maturational stages of neurons in culture that can be used to more successfully plan various types of in vitro experimentation.

The corticostriatal system in dissociated cell culture.

The sparse connectivity within the striatum in vivo makes the investigation of individual corticostriatal synapses very difficult. Most studies of the corticostriatal input have been done using electrical stimulation under conditions where it is hard to identify the precise origin of the cortical input. We have employed an in vitro dissociated cell culture system that allows the identification of individual corticostriatal pairs and have been developing methods to study individual neuron inputs to striatal neurons. In mixed corticostriatal cultures, neurons had resting activity similar to the system in vivo. Up/down states were obvious and seemed to encompass the entire culture. Mixed cultures of cortical neurons from transgenic mice expressing green fluorescent protein with striatal neurons from wild-type mice of the same developmental stage allowed visual identification of individual candidate corticostriatal pairs. Recordings were performed between 12 and 37 days in vitro (DIV). To investigate synaptic connections we recorded from 69 corticostriatal pairs of which 44 were connected in one direction and 25 reciprocally. Of these connections 41 were corticostriatal (nine inhibitory) and 53 striatocortical (all inhibitory). The observed excitatory responses were of variable amplitude (-10 to -370 pA, n = 32). We found the connections very secure - with negligible failures on repeated stimulation (approximately 1 Hz) of the cortical neuron. Inhibitory corticostriatal responses were also observed (-13 to -314 pA, n = 9). Possibly due to the mixed type of culture we found an inhibitory striatocortical response (-14 to -598 pA, n = 53). We are now recording from neurons in separate compartments to more closely emulate neuroanatomical conditions but still with the possibility of the easier identification of the connectivity.

Functional anatomy: dynamic States in Basal Ganglia circuits.

The most appealing models of how the basal ganglia function propose distributed patterns of cortical activity selectively interacting with striatal networks to yield the execution of context-dependent movements. If movement is encoded by patterns of activity then these may be disrupted by influences at once more subtle and more devastating than the increase or decrease of neuronal firing that dominate the usual models of the circuit. In the absence of dopamine the compositional capabilities of cell assemblies in the network could be disrupted by the generation of dominant synchronous activity that engages most of the system. Experimental evidence about Parkinson's disease suggests that dopamine loss produces abnormal patterns of activity in different nuclei. For example, increased oscillatory activity arises in the GPe, GPi, and STN and is reflected as increased cortical beta frequency coherence disrupting the ability to produce motor sequences. When the idea of deep brain stimulation was proposed - it was supported by the information that lesions of the subthalamus reversed the effects of damage to the dopamine input to the system. However, it seems increasingly unlikely that the stimulation acts by silencing the nucleus as was at first proposed. Perhaps the increased cortical beta activity caused by the lack of dopamine could have disabled the patterning of network activity. Stimulation of the subthalamic nucleus disrupts the on-going cortical rhythms. Subsequently asynchronous firing is reinstated and striatal cell assemblies and the whole basal ganglia circuit engage in a more normal pattern of activity. We will review the different variables involved in the generation of sequential activity patterns, integrate our data on deep brain stimulation and network population dynamics, and thus provide a novel interpretation of functional aspects of basal ganglia circuitry.

Slowly progressive dopamine cell loss--a model on which to test neuroprotective strategies for Parkinson's disease?

Making animal models of human disease is a very flawed process. Aspects of the disease can be imitated but models do not necessarily give reliable leads for treatment strategies. When Ungerstedt in Sweden first described the 6-hydroxydopamine (6-OHDA) treated rat model of Parkinson's disease /89/ we knew that the symptoms would not map readily to those of the human disease--rats have four legs after all. On the other hand, the neuropathology looked exactly like end-stage Parkinsonian pathology. That remained true even as we explored other types of neuropathology in the rats /24,43-46,80/. Many of today's treatments for Parkinsonism are developed from pharmacological studies on that model of rats with a chemically induced lesion. However, the 6-OHDA model does not address the important issue of a cure for the disease. The triggers and the time-course of dopamine (DA) cell death in rats are known for nearly every disease model - but for the human disease there is no equivalent knowledge. In the human, the neurons have been dying for a considerable time before the symptoms become obvious and they go on dying even with adequate symptomatic relief /94/, but after intracerebral administration of 6-OHDA to an animal the cells die quickly; all cells are destroyed in less than 5 days /42,88,89/. Thus, we were interested in developing an animal model of DA cell death with a slower time-course. After ibotenic acid injections into rat globus pallidus (GP), DA cells are lost from the ipsilateral substantia nigra over the slower time scale of about six weeks. This time scale has allowed us to test some interventions to prevent the cells from dying. Although some attempts have succeeded, cell death is prevented only for three weeks -beyond that treatments fail and DA cells die. At the moment, this model has at least opened a window into causes of neuronal death in a slower time scale /94/ than previous rodent models.

Dealing with the devil in the detail - some thoughts about the next model of the basal ganglia.

Any simplified description of a brain system that survives 20 years is bound to have some problems. Twenty years of research should have added details that were not known when the now classical description of the basal ganglia circuitry was new. Some examples of such new information about the parts of the original model include: All of these details make it difficult to cling to the model as it was presented, but which of the new information will support a new synthesis? Do we have a coherent replacement for those powerfully heuristic ideas put forward by Albin Young and Penny in 1989? Perhaps in the quantitative anatomical detail there is a plan that may support a new theory. If all of the cortex is not accessible to every striatal cell. If dopamine influences many spines rather than the one it contacts. If we stop looking for a mechanical control system for movement in the basal ganglia and begin to investigate the predictive properties of 'reinforcement learning'. Then maybe a new functional model will emerge that is less tied to anatomical and physiological simplification, but perhaps will still allow us to engineer therapeutic strategies to help patients with movement disorders.

Evaluación del efecto de un aceite de palma parcialmente refinado con un alto contenido en micronutrientes sobre el perfil lipídico de ratas / Effect of partially refined palm oil in lipid profile in rats

El aceite de palma es rico en carotenos, tocoferoles y tocotrienoles. El proceso de refinación para consumo humano, produce algunas alteraciones de sus propiedades. El objetivo de este estudio fue evaluar el efecto de un aceite de palma parcialmente refinado, blanqueado y desodorizado (RBD rojo) sobre el perfil lipídico y niveles de vitamina A (retinol) y E (a tocoferol) en 3 grupos de ratas: B (alimento comercial para animales de laboratorio: ST + 5 por ciento de polvo de yema de huevo), C (ST + 5 por ciento de polvo de yema de huevo + 14 RBD rojo) ambos hiperlipidemia inducida y D (ST + 14 por ciento RBD rojo) frente a un control (A) durante 35 días. El RBD rojo indujo una disminución significativa de CT en los grupos C y D (81 ± 11 mg/dL y 77 ± 7 mg/dL, respectivamente) al compararlos con el grupo control (99 ± 11 mg/dL) para el día 35 de experimentación; así como un incremento del HDL-C en grupo C (53 ± 4 mg/dL) y D (53 ± 5 mg/dL) cuando se compararon con el grupo B (44 ± 3 mg/dL) el cual recibió sólo PH, resultando una menor relación CT/HDL (1,5 ± 0,1). Además en los grupos C y D aumentaron significativamente (p £ 0,05) las concentraciones séricas de retinol (26 ± 5 µg/dL y 58 ± 18 µg/dL) y a tocoferol (165 ± 58 µg/dL y 445 ± 65 µg/dL) respectivamente. Estos resultados permiten concluir que la suplementación con RBD rojo disminuye el CT mejorando la relación CT/HDL. La presencia de ácidos grasos monoinsaturados (oleico) y las altas concentraciones de a-tocoferol y retinol, en el aceite de palma RBD rojo utilizado, incidieron favorablemente sobre el perfil lipídico de las ratas con hiperlipidemia inducida.